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Biomedical subjects

K Hiromi

Publications and source records attributed to K Hiromi.

At least 109 records · Page 6Linked to original sources
PubMed1976

Kinetic study of lipoxygenase-hydroperoxylinoleic acid interaction.

Interaction of lipoxygenase with hydroperoxylinoleic acid, which is the product of this enzyme reaction and acts as an activator, was studied kinetically by the fluorescence stopped-flow method. The kinetic features are consistent with a two-step mechanism involving a fast bimolecular association process followed by a slow unimolecular process. The dissociation constant of the bimolecular process was 3 (+/-2) - 10(-5) M, which was appreciably dependent on temperature and pH, in contrast to the rate constant of the latter process. The enthalpy and the entropy of activation for the unimolecular process were estimated to be 21 kcal/mol and 20 e.u., respectively. The pH dependence of the rate constant indicated that an ionizable group with pK of about 8.6 is involved in the interaction. Linoleic acid, the substrate of lipoxygenase, and oleic acid inhibited the interaction between the lipoxygenase and the hydroperoxylinoleic acid by reducing the rate. A series of saturated monohydric alcohols also reduced the rate of the interaction as the chain length of the alcohols increases, though methanol and ethanol increased the rate of the interaction.

Fatty Alcohols↗
PubMed1976

Studies on the subsite structure of amylases. IV. Tryptophan residues of glucoamylase from Rhizopus niveus studied by chemical modification with N-bromosuccinimide.

Chemical modification of glucoamylase [EC 3.2.1.3] from Rhizopus niveus by N-bromosuccinimide was carried out to investigate the role of tryptophan residues in the enzyme-catalyzed reaction and their location in the enzyme subsites. Of the ten tryptophan residues of the enzyme four could be modified. The two more reactive residues were confirmed not be essential for the catalytic activity for the hydrolysis of maltodextrin and phenyl alpha-maltoside. Complete loss of the catalytic activity, however, was brought about by modifying the two less reactive residues, and the modification of these residues was prevented by the substrates. The characteristic difference spectrum produced by maltose (7) disappeared in parallel with the loss of the catalytic activity. These results suggest that the tryptophan residue(s) responsible for the maltose-induced difference spectrum may be located at one of the subsite near the catalytic site and plays an important role in the catalytic activity of the enzyme.

Binding Sites↗
PubMed1976

Interaction of cyclodextrins with fluorescent probes and its application to kinetic studies of amylase.

It was found that 6-p-toluidinylnaphthalene-2-sulfonate (TNS) showed pronounced fluorescence enhancement when it was added to alpha-, beta-, and gamma-cyclodextrin solutions. 2. The following results were obtained by quantitative study of the interactions of three kinds of cyclodextrins with TNS by following TNS fluorescence at pH5.3. and 25 degrees. i) alpha-Cyclodextrin forms a l : l complex with TNS. ii) beta- and gamma-Cyclodextrins form 1 : 1 and also 2 : 1 complexes; in the latter two cyclodextrin molecules bind to one TNS molecule. iii) The dissociation constants of cyclodextrin-TNS complexes were determined to be 54.9 mM for alpha-cyclodextrin, 0.65 mM for beta-cyclodextrin and 0.66 mM for gamma-cyclodextrin in the 1 : 1 complex, and the secondary dissociation constants in the 2 : 1 complex were 71.4 mM for beta-cyclodextrin in the 1 : 1 complex, and the secondary dissociation constants in the 2 : 1 complex were 71.4 mM for beta-cyclodextrin and 32.6 mM for gamma-cyclodextrin. iv)...

Amylases↗
PubMed1976

Studies on the subsite structure of amylases. III. Inhibition by gluconolactone of the hydrolysis of maltodextrin catalyzed by glucoamylase from Rhizopus niveus.

Inhibition by gluconic acid-1 : 5-lactone (gluconolactone) and phenyl alpha-glucoside of the hydrolysis of maltodextrin catalyzed by glucoamylase [EC 3.2.1.3] from Rhizopus niveus was investigated in relation to the subsite structure of the enzyme. Inhibition by gluconolactone was of the mixed type, whereas that by phenyl alpha-glucoside was purely competitive. These inhibition types were consistent with a theoretical prediction based on the assumption that gluconolactone and phenyl alpha-glucoside bind mainly to Subsites 1 and 2, respectively. The inhibitor constant of gluconolatone was determined to be 1.5 mM, which is in good agreement with the dissociation constant estimated by difference spectrophotometry (1.5 mM) (Ohnishi, M, et al. (1975) J. Biochem, 77, 695-703).

Amylases↗
PubMed1976

Determination of the best-fit values of kinetic parameters of the Michaelis-Menten equation by the method of least squares with the Taylor expansion.

The best-fit values of the Michaelis constant (Km) and the maximum velocity (V) in the Michaelis-Menten equation can be obtained by the method of least squares with the Taylor expansion for the sum of squares of the absolute residual, i.e., the difference between the observed velocity and the corresponding velocity by calculation. This method makes it possible to determine the values of Km and V not in a trial-and-error manner but in a deductive and unique manner after some iterative procedures starting from arbitrary approximate values of Km and V. These values can be said to be uniquely determined for a set of data as the finally converged values are no longer dependent upon the initial approximate values of Km and V. It is also very important to obtain initial approximate values of parameters for the application of the method described above. A simple method is proposed to estimate the approximate values of parameters involved in fractional functions. The method of rearrangement after canceling of denominator of a fractional function can be utilized to obtain approximate values, not only for cases of two unknown parameters such as the Michaelis-Menten equation, but also for cases with more than two unknowns.

AMP Deaminase↗
PubMed1976

Quantitative analysis of the action of Taka-amylase A on maltotriose.

The action of Taka-amylase A from Asp. oryzae was studied quantitatively by the product analysis method using unlabeled maltotriose and maltotriose labeled at the reducing end as substrates. It was found that the ratio of the unlabeled products, maltose (G2) and glucose (G1) exceeded unity, and that the ratio of the labeled products, G2/G1 was strongly dependent on the initial substrate concentrations. The results can only be explained by a transglycosylation or condensation mechanism or both. Analysis of maltose labeled at the nonreducing or reducing end reveal that the ratio of the transglycosylation to the condensation mechanism was about 20:1 with about 80 mM maltotriose. A computer simulation was made on a reaction scheme involving the termolecular-shifted complex, transglycosylation and condensation besides hydrolysis, by using reported subsite affinities as modified by the authors. The simulation reproduced the experimental results satisfactorily.

Amylases↗
PubMed1976

A method for the deductive and unique determination of the values of three parameters involved in fractional functions applicable to relaxation kinetics.

A novel method is proposed to determine deductively and uniquely the values of three parameters, a, b, and c in a fractional function of the form, y=a+bx/(c+x) where x and y are experimentally obtainable variables. This type of equation is frequently encountered in chemistry and biochemistry involving relaxation kinetics. The method of least squares with the Taylor expansion is employed for direct curve fitting of observed data to the fractional function. Approximate values of the parameters, which are always necessary prior to commending the above procedure, can be obtained by the method of rearrangement after canceling the denominator of fractional functions. This procedure is very simple, but very effective for estimating provisional values of the parameters. Deductive and unique determination of the parameters involved in the fractional function shown above can be accomplished for the first time by the combination of these two procedures. This method is extended to include the analysis of relaxation kinetic data such as those of temperature-jump method where the determination of equilibrium concentrations of reactants in addition to the three parameters is also necessary.

Acridines↗
PubMed1975

Studies on the interaction between Streptomyces pepsin inhibitor and several acid proteinases by means of a zinc(II)-dye complex as a probe.

The zinc(II) complex of pyridine-2-azo-p-dimethylaniline is bound to several acid proteinases, at pH 5.0, accompanied by a change is the visible absorption spectrum. Streptomyces pepsin inhibitor, which was discovered by Satoi and Murao (Satoi, S. and Murao, S. (1970) Agric. Biol. Chem. 34, 1265-1267 and Satoi, S. and Murao, S. (1971) Agric. Biol. Chem. 35, 1482-1487), is also bound to acid proteinases. Spectrophotometric studies with ten acid proteinases from different sources have revealed that in several acid proteinases, zinc(II)-pyridine-2-azo-p-dimethylaniline is released from the enzyme by the inhibitor, while some acid proteinase forms a quaternary complex, zinc(II)-pyridine-2-azo-p-dimethylaniline-inhibitor-enzyme. It is speculated that zinc(II)-pyridine-2-azo-p-dimethylaniline is bound to two catalytic carboxylate groups in the active site of the acid proteinases and the inhibitor is bound mainly to the substrate-binding site of the enzymes. The binding of the inhibitor may overlap the catalytic site completely or partially. The degree of overlapping is characteristic of the kind of acid proteinases.

Aspergillus↗
PubMed1975

Studies on the subsite structure of amylases. I. Interaction of glucoamylase with substrate and analogues studied by difference-spectrophotometry.

Studies were made on the ultraviolet difference-spectra of glucoamylase from Rhizopus niveus [EC 3.2.1.3] specifically produced by the substrate maltose and the inhibitors, glucose, glucono-1: 5-lactone (gluconolactone), methyl beta-D-glucoside, cellubiose, and cyclohexa-, and cyclohepta-amyloses. Of these, maltose and gluconolactone produced characteristic difference spectra with a trough near 300 nm. Based on studies with a model compound for a tryptophan residue, Ac-Trp, this trough was attributed to the effect of a negative charge upon the tryptophan residue. From the concentration dependency of the difference spectra, the dissociation constants of the complexes between the enzyme and maltose, glucose, and gluconolactone were evaluated to be 1.2 mM, 51 mM, and 1.5 mM, respectively. These values are in good agreement with the values of Km or K1 obtained from the steady-state kinetics. The difference-spectrophotometric data suggested that referring to the values of subsite affinities of glucoamylase, maltose, and gluconolactone occupy mainly Subsite 1, where the non-reducing-end glucose residue of a substrate is bound in a productive form and that a tryptophan residue with shows a trough near 300 nm in difference spectra is located in this subsite.

Amino Acids↗
PubMed1975

Temperature-jump studies on benzeneboronic acid-serine protease interactions. Subtilisin and alpha-chymotrypsin.

The interactions of benzeneboronic acid (BBA) as a transition state analog with subtilisin (EC 3.4.21.4) and with alpha-chymotrypsin (EC 3.4.21.1) were investigated kinetically by the temperature-jump method using pH indicators. For both enzymes, the concentration dependence of the relaxation time was consistent with a two-step mechanism involving a fast bimolecular association followed by a slow, unimolecular process. The possibility of a trigonal-tetrahedral interconversion of BBA at the active site of the enzyme is discussed.

Benzene↗
PubMed1975

The pH jump study of enzyme proteins. I. Liquefying alpha-amylase from Bacillus subtilis.

Rapid conformational changes due to pH jump were studied kinetically at 25 degrees mainly by the stopped-flow method using liquefying alpha-amylase from Bacillus subtilis [EC 3.2.1-.1, liquefying]. First, the conformational change due to a pH jump produced by mixing with alkali was monitored as a function of time at 245 nm through the ionization of phenolic hydroxyl groups of tyrosine residues which were originally buried and finally become exposed due to the pH jump. Three distinct phases of conformational change were clearly recognized by this method by varying the final pH values. Each phase involved the exposure of an essentially definite number of tyrosine residues, whose rate constant was crucially dependent on pH. Second, these phases of conformational change were subjected to examination in terms of the optical rotation change at 411 nm and the reversibility upon reverse pH jump with respect to conformational reconstitution, as observed through the protonation ofphenolic hydroxyl groups of ionized tyrosine residues and the enzyme activity. The first phase, which occurs above pH 12.5, involves no change in the optical rotation and is reversible as observed by the above two monitoring methods. In contrast, the other two phases, which are observed above pH 12.7, are accompanied by an optical rotation change and no appreciable reversibility was detected by these methods.

Amylases↗
PubMed1975

Substrate concentration dependence of the rate of maltose hydrolysis by saccharifying alpha-amylase from B. subtilis.

1. The action of saccharifying alpha-amylase from B. subtilis [EC 3.2.1.1] on eq- (the equilibrium mixture of alpha- and beta-forms) and beta-maltose was studied at pH 5.4 and 25 degrees. 2. The plots of initial velocity versus substrate concentration showed remarkable sigmoidal curves for both substrates. 3. The value of Hill's coefficient for eq- and beta-maltose were determined to be 3.3 and 3.8, respectively.

Amylases↗
PubMed1975

Studies on the subsite structure of amylases. II. Difference-spectrophotometric studies on the interaction of maltotriose with liquefying alpha-amylase from Bacillus subtilis.

The difference spectra of liquefying alpha-amylase (EC 3.2.1.1) from B. subtilis upon the addition of a slowly reacting substrate, maltotriose, were measured to investigate specific binding of the substrate to the enzyme. The spectra produced by maltotriose were attributed to one tryptophan and one tyrosine residues on the basis of analysis of their shape and magnitude. From the dependence of the difference absorption upon the concentration of maltotriose, the dissociation constant of the maltotriose-enzyme complex was determined to be 170(+/- 20) mM, which is in good agreement with the Michaelis constant, Km obtained from the steady-state kinetics. The difference spectrum characteristic of a tryptophan residue was significantly decreased by the chemical modification of a trytophan residue with N-bromosuccinimide.

Amylases↗
PubMed1975

Elementary processes in the interaction of serine protease with a possible transition state analog. Subtillisin-benzeneboronic acid system.

The interaction of benzeneboronic acid(BBA), a possible transition state analog, with subtilisin BPN' [EC 3.4.21.14] was studied by the temperature-jump method at various pH's, temperatures and in D2O as well as H2O. From analysis of the concentration dependence of the relaxation times, it was suggested that the subtillsin-BBA interactions consist of at least two elementary steps, a fast bimolecular association followed by a slow unimolecular process. Similar concentration dependence was observed at pH 6.1-6.7 at 25degrees. However, in D2O the reciprocal relaxation times generally decreased compared to those in H2O and became concentration-independent below pD 6.5. The relaxation times were influenced considerably by the temperature. From these results, the slow unimolecular process was assigned to the trigonal-tetrahedral interconversion of BBA at the active site of the enzyme.

Benzene↗
PubMed1975

Quantitative determination of anomeric forms of sugar produced by amylases. V. Anomeric forms of maltose produced in the hydrolytic reaction of substituted phenyl alpha-maltosides catalyzed by saccharifying alpha-amylase from B. subtilis.

1. Hydrolyses of phenyl alpha-maltoside and its derivatives with various substituents (p-NO2, p-C1, p-CH3, p-C2H5, and p-C(CH3)3) catalyzed by saccharifying alpha-amylase from B. subtilis3 [EC 3.2.1.1] were studied under conditions such that the products were only maltose and the corresponding phenols (1), in order to determine quantitatively the anomeric form of the sugar produced from each substrate. 2. At the optimum pH of this enzyme (pH-5.4), maltose released from all the substituted substrates studied was entirely in the beta-form. These results are in remarkable contrast to the previous finding that alpha-maltose is exclusively produced from unsubstituted phenyl alpha-maltoside by this enzyme (2). 3. At pH 6.18 and 6.73, maltose produced from unsubstituted phenyl alpha-maltoside (øM) or p-tert-butylphenyl alpha-maltoside (PTBøM) was a mixture of alpha- and beta-anomers, the ratio being dependent on pH as follows: For øM, the percentage of alpha-anomer was 100% (pH 5.4), 80 (pH 6.18), and 55% (pH 6.73), whereas for PTBøM, the percentage of beta-anomer was 100% (pH 5.4), 75% (pH 6.18), and 60% (pH 6.73).

Amylases↗